The present invention relates generally to an apparatus and method for spectroscopic analysis, and more particularly to an apparatus and method for real-time generation and collection of fluorescence, reflection, scattering, and absorption information from a tissue sample to which multiple excitation wavelengths are sequentially applied.
Attempts at in situ real-time diagnostics for complex biological media, have been only marginally successful because of limitations in the spectroscopic techniques that are applicable. Conventional fluorescence spectroscopy is generally unable to resolve differences among similar biological tissue samples (or subtle differences in a given tissue sample) and has generally not proven reliable in detecting malignancy except with the aid of drugs such as hematoporphyrin derivatives which are used as targeting fluorescers (see, e.g., D. Kessel, "Tumor Localization And Photosensitization By Derivatives of Hematoporphyrin: A Review," IEEE J. Quantum Electron. QE-23, 1718 (1987)). This is because broad-band fluorescence from one group of chromophores can mask the emission from others, and if a different excitation wavelength is chosen to avoid that problem, other information is lost by the fact that only some types of the fluorescers are then excited. Recently researchers have demonstrated an improved technique for the fluorescence spectroscopy of tissue, called "fluorescence contour mapping" by some (see, e.g., R. Richards-Kortum et al., "Fluorescence Contour Mapping: Applications To Differentiation Of Normal And Pathological Human Tissues," Program of the Conference on Lasers and Electro-Optics 1989, 1989 Technical Digest Series 11, 140). In this technique, fluorescence spectra are obtained for many excitation wavelengths. The data are obtained using commercial instrumentation without fiber-optic transport of the light, and are presented in a two-dimensional (topographical) contour plot in which the fluorescence wavelength is plotted against the excitation wavelength for a series of intensity levels. This approach has proved capable of detecting subtle differences between healthy tissue and that exhibiting pre-malignancy changes. The principal disadvantages of the procedure, however, are that a single plot requires several hours of data gathering (thus eliminating its usefulness for real-time diagnostics) and that tissue is not examined in situ.
Excitation-Emission Matrix Fluorescence is a similar well-known technique from analytical chemistry for analyzing mixtures of organics. In its most usual form, this technique is slow, but precise. See, e.g., I. M. Warner, G. Patonay, and M. P. Thomas, "Multidimensional Luminescence Measurements," Analytical Chem. 57, 463A (1985), which reviews all fluorescence techniques used in analytical chemistry. A less precise, but rapid, form of Excitation-Emission Matrix Fluorescence has been developed. See, e.g., I. M. Warner, M. P. Fogarty, and D. C. Shelly, "Design Considerations For A Two-Dimensional Rapid Scanning Fluorimeter," Analytica Chemica Acta 109, 361 (1979) which is the pioneering work on dispersed-excitation-spot Excitation-Emission Matrix Fluorescence. This technique requires a large spot (several cm) containing a dispersed range of excitation wavelengths. Indeed, it is mentioned in the first Warner et al. reference that analysis speed can be obtained only at the cost of a large sampling area. Such large sampling areas are unsuitable for medical diagnostics because of the sub-mm-sc inhomogeneity of real biological samples. An apparatus for the Excitation-Emission Matrix Fluorescence analysis of remote samples, in which the excitation and emissions are delivered and gathered by means of optical fibers is described in J. B. Zung, R. L. Woodlee, M-R. S. Fuh, and I. M. Warner, "Recent Developments and Applications of Multidimensional Fluorescence Spectroscopy," SPIE 1054, Proceedings of Fluorescence Detection III (1989), p. 69; J. B. Zung, R. L. Woodlee, M-R. S. Fuh, and I. M. Warner, "Fiber Optic Based Multidimensional Fluorometer for Studies of Marine Pollutants," SPIE 990, Proceedings of Chemical, Biochemical, and Environmental Applications of Fibers (1988), p. 49; J. B. Zung, M-R. S. Fuh, and I. M. Warner, "Design and Characterization of a Fiber Optic-Based Fluorimeter," Anal. Chim. Acta 224, 235 (1989). Optical fibers are employed in an apparatus which has similarities with the present invention to access remote samples. These references teach the rejection of scattered radiation from samples by the light gathering apparatus, and in fact describe the deployment of the light delivery and light collection fibers in such a manner that scattering is minimized.
Single-excitation-wavelength fluorescence spectra are currently being widely studied for some specific analyses tissue partly as a result of increased interest in laser angioplasty (see, e.g., R. Richards-Kortum et al., "A Model For The Extraction Of Diagnostic Information From Laser Induced Fluorescence Spectra Of Human Artery Wall," Spectrochimica Acta 45A, 87 (1989). Other applications of single-spectrum analysis are illustrated in: R. M. Cothren et al., "Gastrointestinal Tissue Diagnosis By Laser-Induced Fluorescence Spectroscopy At Endoscopy," Gastrointestinal Endoscopy 36, 105 (1990); R. R. Alfano, A. Pradhan, G. C. Tang, and S. J. Wahl, "Optical Spectroscopic Diagnosis Of Cancer And Normal Breast Tissues," J. Opt. Soc. Am. B 6, 1015 (1989); and R. R. Alfano and M. A. Alfano, "Method For Detecting Cancerous Tissue Using Visible Native Luminescence," U.S. Pat. No. 4,930,516). Therein, the tissue to be examined is excited with a beam of monochromatic light, after a determination of the wavelength(s) at which maximum intensity of fluorescence occurs, and the fluorescence measured as a function of wavelength. A comparison of the spectrum of the excited tissues under investigation with the spectrum of known noncancerous tissue, allows the determination of the carcinomatoid status thereof, albeit with variable reliability. It is important to recognize that biological tissue is a form of matter unlike those (liquid, gas, or solid) normally encountered in analytical chemistry. With the exception of solid forms (bones, teeth, hair, etc.) tissue is usually composed of variously encapsulated liquids or semi-liquids. Not only is tissue inhomogeneous on a small scale, but it has a great deal of physical structure to it. Therefore, fluorescence measurement alone is an incomplete diagnostic for tissue. Fluorescence can reveal much about the chemical makeup of the tissue as a whole, but provides nothing about its structure. However, other optical properties of tissue such as reflection, absorption, and scattering may assist in more detailed analyses. Optical scattering (in which cellular structures divert the photon in a different direction without changing its energy) is very dependent on the tissue structure and, accordingly, quite sensitive to changes therein that accompany the development of cancer. It is usually such changes that a pathologist detects by eye when analyzing biopsy samples. The most sensitive and complete optical diagnostic for detecting the chemical and structural changes in the development of cancerous tissue would therefore be one that could detect all the possible optical responses of tissue.
Accordingly, it is an object of the present invention to provide an apparatus and method for real-time fluorescence analysis of tissue at multiple excitation wavelengths with a simultaneous measure of the absorption and scattering as a function of wavelength.
Another object of the invention is to provide an apparatus and method for identification of cancerous tissue either in vitro or in vivo in real time with high reliability.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.